HIV latency is emerging as barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill', reactivation of latent virus using non-targeted small molecules. However, significant hurdles will have to be overcome for this approach to be successful. These include stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upn re- activation. In addition to these limitations, most of what we know about latency is derived from the study of latency in peripheral blood mononuclear cells. Using a newly developed dual fluorescence reporter HIV, we have observed that latency was significantly more prevalent in CD4 T cells isolated from oral lymphoid tissues (tonsil) than in CD4 T cells from peripheral blood or spleen. In addition, latency in CD4 T cells from oral lymphoid tissues was strongly dependent on the presence of non-CD4 T cells. Thus, the biology of latency and its reactivation may differ in primary oral lymphoid tissues when compared to other lymphoid tissues. If so, therapeutic interventions might need to be modified to account for the unique characteristics of latency in oral lymphoid tissues. We propose to use a multi-pronged, systems-biology strategy to further define HIV latency and its mechanism in primary oral lymphoid tissues. We will use our new HIV-1 reporter to identify, quantify, and purify latently infected cells in their native state earl after infection of tonsil CD4 T cells. Purified latently infected cells will be characterized usingthe following tools: 1. CyTOF single-cell analysis of cell-surface markers, T-cell signaling networks, and T-cell effector function; 2. RNAseq and microRNAseq analysis of the cellular transcriptome; 3. Mechanistic studies using RNP-Cas9 gene interference to identify key regulators of HIV latency in primary oral lymphoid tissues. In addition, we will define the relative contribution of intercellular interactions and secreted factors in the ability of non-CD4 T cells to influence HIV latency in CD4 T cells in oral lymphoid tissues. By combining our dual-fluorescence latency model with powerful systems biology techniques and the tonsil HLAC system, we expect to significantly increase our understanding of HIV latency and to identify potential new therapeutic opportunities.